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Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for
Daniela Schulz1,2, Laura Feulner1,2, Dominique Santos Rubenich1,2,3
1Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Germany.
Abstract:
The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.
Insights
Programmed cell death 1 ligand 1 (PD-L1) has novel nuclear functions in head and neck cancer cells, independent of immune signaling. These findings reveal complex PD-L1 regulation and impact immune checkpoint inhibitor therapy efficacy.
Area of Science:
- Oncology
- Immunology
- Cell Biology
Background:
- The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) pathway is crucial for immune suppression.
- Emerging evidence suggests PD-L1 has functions beyond its role as a ligand, particularly within tumor cells.
- PD-L1 localization is not restricted to the cell membrane, with presence in various subcellular compartments.
Purpose of the Study:
- To investigate the non-canonical cellular functions and subcellular localization of PD-L1 in head and neck cancer (HNC).
- To identify novel PD-L1 variants and their potential roles within tumor cells.
- To explore the implications of these findings for immune checkpoint inhibitor (ICI) therapy.
Main Methods:
- Subcellular fractionation and Western blotting to detect PD-L1 in HNC cell lines and primary tumors.
- Immunofluorescence staining to assess PD-L1 expression during the cell cycle.
- Proximity ligation assays and mass spectrometry to identify interacting proteins and pathways.
- Gene ontology analysis to understand the functional implications of nuclear PD-L1 interactions.
Main Results:
- PD-L1 was detected in the nucleus of HNC cells, alongside previously unidentified variants (~70 kDa and >150 kDa).
- Nuclear PD-L1 variant expression demonstrated cell-cycle dependence.
- Vimentin was identified as a potential mediator of nuclear PD-L1 transport.
- Nuclear PD-L1 interacts with proteins involved in DNA remodeling and mRNA splicing.
Conclusions:
- PD-L1 exhibits complex, cell-intrinsic functions within HNC cells, independent of immune regulation.
- The discovery of nuclear PD-L1 variants and their interactions suggests novel roles in tumor biology.
- These findings have significant implications for understanding and optimizing the efficacy of ICI therapies.
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